Sinomonas terrae sp. nov., Isolated from an Agricultural Soil

While searching for the bacteria which are responsible for degradation of pesticide in soybean field soil, a novel bacterial strain, designated 5-5T, was isolated. The cells of the strain were Gram-staining-positive, aerobic and non-motile rods. Growth occurred at 10-42°C (optimum, 30°C), pH 5.5-9.0 (optimum, pH 7.0-7.5), and 0-2% (w/v) NaCl (optimum, 1%). The predominant fatty acids were C15:0 anteiso, C17:0 anteiso, and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The predominant menaquinone was MK-9 (H2). Diphosphatidylglycerol, glycolipids, phosphatidylinositol, and phosphatidylglycerol were the major polar lipids. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain 5-5T is a member of the genus Sinomonas and its closest relative is Sinomonas humi MUSC 117T, sharing a genetic similarity of 98.4%. The draft genome of strain 5-5T was 4,727,205 bp long with an N50 contig of 4,464,284 bp. Genomic DNA G+C content of strain 5-5T was 68.0 mol%. The average nucleotide identity (ANI) values between strain 5-5T and its closest strains S. humi MUSC 117T and S. susongensis A31T were 87.0, and 84.3 % respectively. In silico DNA-DNA hybridization values between strain 5-5T and its closest strains S. humi MUSC 117T and S. susongensis A31T were 32.5% and 27.9% respectively. Based on the ANI and in silico DNA-DNA hybridization analyses, the 5-5T strain was considered as novel species belonging to the genus Sinomonas. On the basis of the results from phenotypic, genotypic and chemotaxonomic analyses, strain 5-5T represents a novel species of the genus Sinomonas, for which the name Sinomonas terrae sp. nov. is proposed. The type strain is 5-5T (=KCTC 49650T=NBRC 115790T).


Phylogenetic Analysis
To amplify the partial 16S rRNA gene of strain 5-5 T , colony PCR was conducted using the forward primer 27F (5-AGAGTTTGATCMTGGCTCAG-3) and reverse primer 1492R (5-GGYTACCTTGTTACGACTT-3), which were previously described [10]. Following the PCR amplification of the partial 16S rRNA gene of strain 5-5 T , the PCR product was purified and sequenced using the 785F and 907R universal bacterial primers. The resulting sequence data was compiled using SeqMan software, which is developed by DNASTAR in the USA. Specifically, the software was used to generate an almost complete sequence of the 16S rRNA gene. To identify the closest phylogenetic species of strain 5-5 T , the 16S rRNA gene sequence was analyzed using the EzBioCloud server (http://ezbiocloud.net) [11]. To perform the phylogenetic analysis, 16S rRNA gene sequences of the closest relatives were obtained from the GenBank database. The obtained 16S rRNA gene sequences of the related bacteria were aligned through multiple alignment using SILVA alignment tool according to the SILVA seed alignment [12]. To construct the phylogenetic trees, three algorithms were used: neighbor-joining [13], maximumparsimony [14], and maximum-likelihood [14], using the software MEGA7 [15]. Kimura's two-parameter model was utilized to compute the evolutionary distances between the aligned 16S rRNA gene sequences [16]. Bootstrap analysis was performed using 1,000 replications [17].

Whole Genome Sequencing, Assembly, and Annotation
For whole genome sequencing, genomic DNA of strain 5-5 T was extracted using a Maxwell® Prokaryote/ Eukaryote SEV DNA Purification Kit (Promega) according to the manufacturer's procedure. Whole genome shotgun sequencing was performed using the Illumina HiSeq2500 platform by Macrogen in South Korea. The sequencing data was then assembled using SPAdes version 3.14 [18], and the resulting genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [19]. The DNA G+C content of strain 5-5 T was determined by analyzing the whole genome sequence. To determine the genome-based relatedness between strain 5-5 T and closest type strains, the ANI calculator [20] was used to determine the Average Nucleotide Identity (ANI), and the Genome-to-Genome Distance Calculator (GGDC 2.1; https://ggdc.dsmz.de/ggdc.php) [21] was utilized for in silico DNA-DNA hybridization (dDDH) value calculations. These values are commonly used to estimate the genetic relatedness between bacterial strains at the species level.

Phenotypic Characterization
Cellular morphology of strain 5-5 T was determined using transmission electron microscopy (LIBRA 120; Carl Zeiss, Germany). The cells were grown on R2A agar for 2 days at 30 o C before being examined. The strain 5-5 T was tested for growth on different types of media including Nutrient Agar (NA), Luria-Bertani Agar (LBA), R2A Agar (R2A), Trypticase Soy Agar (TSA), and MacConkey Agar (MCA). All the media were manufactured by BD Difco in the USA. The activities of Catalase and Oxidase of the strain 5-5 T were tested using commercial reagents, specifically the ID Color Catalase Reagent and Oxidase Reagent from bioMérieux, France. The strain 5-5 T was subjected to tests for its ability to grow at various temperatures, pH levels, and NaCl concentrations in R2A broth after 7 days of incubation at 30 o C. To determine the temperature range suitable for growth, the strain was subjected to growth testing at various temperatures ranging from 4 o C to 45 o C. The temperatures tested were 4, 10, 18, 25, 30, 37, 42, and 45 o C. The pH range for growth was determined by adjusting the pH of the R2A broth to 4.0-10.0 with citrate buffer (pH 4.0-5.5), phosphate buffer (pH 6.0-7.5), and Tris buffer (pH 8.0-10.0). Tolerance to NaCl was tested in R2A broth with varying concentrations of NaCl (0-10% w/v). To determine the growth of the strain in anaerobic conditions, an anaerobic growth test was conducted by incubating strain 5-5 T on R2A agar at 30 o C for 14 days using an anaerobic jar and BD GasPak EZ Gas Generating Pouch System. After being incubated for 14 days, the strain's capacity for hydrolyzing casein, starch, DNA, cellulose, and Tween 80 was evaluated according to the Smibert and Krieg's methods [22]. The API ZYM and API 20NE systems (bioMérieux, France) were utilized to test for other physiological and biochemical properties.

Chemotaxonomic Characterization
To examine the cellular fatty acid composition, a fatty acid methyl ester (FAME) analysis was conducted. The strain 5-5 T , Sinomonas humi NBRC 110653 T , Sinomonas susongensis JCM 31752 T , Sinomonas atrocyanea KACC 17386 T , and Sinomonas soli KCTC 19389 T were cultivated on R2A agar at 30 o C for 2 days at the same time. After cultivation, the cellular fatty acids were extracted, methylated, and separated using gas chromatography (model 7890; MIDI, USA) following the Sherlock Microbial Identification System's protocol [23]. The identification and quantification of fatty acid methyl esters were performed using the Sherlock Microbial Identification System's TSBA 6 database (version 6.3). After cultivation on R2A agar, the cells were harvested by centrifugation and subjected to freeze-drying for analysis of isoprenoid quinones. The extraction of isoprenoid quinones was performed using a chloroform/methanol solution (2:1, v/v), followed by purification with a Sep-pak kit (Waters, USA) and subsequent analysis via HPLC, as described by Collins and Jones [24]. The freeze-dried cells were used for extracting polar lipids, which were then separated using two-dimensional TLC based on the method developed by Minnikin et al. [25]. Individual polar lipids were identified by spraying the plates with 10% ethanolic molybdophosphoric acid for total lipids, molybdenum blue for phospholipids, ninhydrin for amino lipids, αnaphthol reagents for glycolipids, and Dragendorff 's reagent for phosphatidylcholine [26].

Phylogenetic Analysis
Following 16S rRNA gene PCR sequencing, strain 5-5 T yielded a 1,460 bp sequence, which was deposited in GenBank under the accession number MT849769. The strain 5-5 T belonged to the class Actinomycetia, order Micrococcales, and family Micrococcaceae. Hihg sequence similarity was observed with S. humi MUSC 117 T , S. susongensis A31 T , S. atrocyanea KCTC 3377 T , and S. soli CW 59 T (98.4, 98.4, dk97.3, and 97.2% sequence similarity, respectively). Based on the threshold value of 98.7% similarity for bacterial species delineation [27], the strain 5-5 T represents a novel species of the genus Sinomonas. Analysis using three different tree-building approaches (neighbor-joining, maximum-parsimony, and maximum-likelihood) indicated that strain 5-5 T was closely related to other members of the Sinomonas genus (Fig. 1). The closely related type strain S. humi NBRC 110653 T , S. susongensis JCM 31752 T , S. atrocyanea KACC 17386 T , and S. soli KCTC 19389 T were chosen as reference strains.

Whole Genome Sequencing, Assembly, and Annotation
The complete genome sequence of strainm5-5 T has been submitted to DDBJ/ENA/GenBank and assigned the accession number JAKZBV000000000. The size of the genome was 4,727,205 bp, with an N50 value of 4,464,284 bp ( Table 1). The genome of the strains consists 3 contigs with genome coverage of 207.86×. The genome included   [20]. In silico DDH values between strain 5-5 T and its closest strains S. humi MUSC 117 T , S. susongensis A31 T , and S. atrocyanea KCTC 3377 T were 32.5%, 27.9%, and 21.7%, respectively. These values were found to be below the threshold of 70% [21]. These results suggested that the strain 5-5 T exhibits species-level differences from other species of the genus Sinomonas.

Phenotypic Characterization
Cells of strain 5-5 T were identified as Gram-staining-positive, non-motile, and aerobic rods with 0.6-0.75 × 2.5-3.0 μm in size. (Fig. 2). The strain formed smooth, convex, circular and creamy-white colonies when grown on R2A agar at 30 o C for 2 days. Enzyme reaction for oxidase was negative and catalase was positive. Strain 5-5 T grew on NA, R2A, LB, TSA, MCA. Strain 5-5 T hydrolyzed starch and Tween 80 but could not hydrolyze casein, DNA, and cellulose. The strain 5-5 T exhibited a growth range between 10-42 o C, pH 5.5-9.0, and 0-2% (w/v) NaCl. The Optimal growth conditions for the strain were observed at 30 o C, pH 7.0-7.5, and 1% (w/v) NaCl. The results of physiological and biochemical features of strain 5-5 T and other Sinomonas species are presented in detail in Table 2.

Table 2. Differential phenotypic characteristics of strain 5-5 T and type strains of closely related species.
C h a r a c t e r i s t i c s 1 2 3 4 5 Assimilation of: predominant polar lipids in the strain 5-5 T are DPG, PG, PI, and GL. These polar lipids are typical for species within the genus Sinomonas. Therefore, the polar lipids pattern support that the strain 5-5 T can be affiliated with the genus Sinomonas. The dominant menaquinone present in the strain was MK-9(H 2 ), which is a typically found in species belonging the genus Sinomonas.
The presented finding suggested that the strain 5-5 T shares some common characteristics with other species of the genus Sinomonas, but also exhibits distinct features that set it apart from them. Therefore, the strain 5-5 T is proposed to be a new species within the genus Sinomonas, and named as Sinomonas terrae sp. nov.